In vivo optical imaging is a powerful methodology allowing users to monitor molecular and functional events noninvasively in a broad array of applications from cancer biology to microbiology using either bioluminescent or fluorescent reporters. The ultimate success and reproducibility of your imaging experiments can be greatly enhanced by following these simple tips.

Choose reporters that maximize signal-to-noise (S:N) ratio. Bioluminescent reporters offer unparalleled S:N ratios. Red-shifted fluorescent dyes and proteins have maximum S:N in the near-infrared end of the spectrum where autofluorescence and attenuation are lowest.

Consider the appropriate control groups and imaging time points necessary. Take a time-zero image of each subject. With fluorescent models, a naïve control is fundamental and will allow for easier differentiation of signal from background, also facilitating advanced background subtraction features such as spectral unmixing.

Use hairless mice or white-furred animals and depilate or shave. Dark pigment in the fur or skin strongly absorbs light. Always remove the fur on and around the areas of the animal that are to be imaged. For quantitative accuracy, continue this practice throughout the study.

Switch to autofluorescence-free mouse diet. Regular mouse diet contains chlorophyll that autofluoresces around 700 nm and can interfere with signal collected from probes with emission in this range.

Closely map the kinetics of your biological model. Bioluminescence requires injection of a substrate. A kinetics curve of light intensity over time should be established for your model to determine the optimal time post-injection to record peak-signal intensity. Firmly adhere to your established imaging time point.

Animal handling can significantly affect kinetics. Anesthesia slows the metabolism thus causing a decrease in body temperature significantly affecting the kinetics of your model. Always administer substrate at the same time point in relation to anesthesia delivery, and keep the animals warm throughout the procedure.

Image in the animal orientation that yields the highest signal intensity. Reporter detection will vary based on animal positioning, which affects the tissue depth. Always place the animal in a position where the source of light (probe) is closest to the detector or, if unknown, image both sides of the animal.

Utilize guards to prevent reflection off of neighboring animals. A significant amount of signal can reflect off neighboring animals and in some cases cause signal contamination. Use guards between animals to prevent this.

One final tip for in vitro imaging: use black well plates. Both white and clear plates reflect light, leading to high background. Furthermore, clear plates allow light contamination into neighboring wells.

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